1. TRANSCRIPTIONAL ACTIVATION AND SWI/SNF-DEPENDENT NUCLEOSOME MOBILIZATION. In collaboration with Dr. Bruce H. Howard (NICHD). Previously, we have mapped nucleosome positions on two genes in yeast, CUP1 and HIS3, using a high-resolution mapping technique called monomer extension. We found that the nucleosome position maps are more complex than anticipated: instead of the unique positions inferred from the classical low-resolution indirect end-labeling technique, we observed clusters of overlapping positions for each nucleosome. A typical cluster has a dominant position with overlapping minor positions, as is generally observed with reconstituted nucleosomes in vitro. In other words, some cells have the nucleosome in one position and others have it in a different position. We suggest that this reflects nucleosome dynamics in cells. How general is this nucleosome position cluster organization - are CUP1 and HIS3 exceptional, or are they typical? The new high-throughput sequencing technology has made it possible to map nucleosomes genome-wide in a relatively simple experiment involving digestion of nuclei with micrococcal nuclease (MNase). We have performed a genome-wide paired-end sequencing study of nucleosomes derived from cells treated with 3-aminotriazole (3AT), which induces Gcn4-dependent genes, and control cells (Cole et al., 2011b). The questions addressed were: (1) How general is the position cluster organization of yeast chromatin? (2) What effect does 3AT induction have on the chromatin structure of activated genes? Analysis of nucleosome positioning on HIS3 confirmed the position cluster organization which we had reported previously for CUP1 and HIS3. Examination of the well-studied PHO5 promoter and the TRP1 gene revealed that both loci have a cluster organization, usually with a clear dominant position, rather than the perfectly positioned nucleosomes inferred from indirect end-labeling. We conclude that each nucleosome may adopt one of several alternative positions within a cluster and that this cluster organization is genome-wide. The implication is that different cells within the population have different chromatin structures. Thus, binding sites for regulatory factors might be nucleosomal in some cells and in the linker in other cells. We propose that the position cluster organization of the yeast genome reflects the presence of alternative nucleosome arrays with the same nucleosome spacing, interspersed by nucleosome-depleted regions. Induction with 3AT results in a major disruption of nucleosome positioning, sometimes with altered nucleosome spacing (Cole et al., 2011b). The most affected genes (50 in all) exhibit a dramatic loss of occupancy over the transcribed region, sometimes extending into neighboring genes. In contrast, nucleosome-depleted promoters are generally unaffected by induction. A small number of genes are repressed by 3AT; these show similar changes in their chromatin structure, but in reverse: loss of nucleosome occupancy is observed in control cells rather than in 3AT-treated cells. Thus, loss of nucleosome occupancy correlates with gene activation. We also discovered that the specialized nucleosome at the centromere is essentially perfectly positioned, unlike canonical nucleosomes, which occupy one of several alternative overlapping positions (Cole et al., 2011a). We attribute the perfect positioning of the centromeric nucleosome to the presence of sequence-specific factors built into the nucleosome. Currently, we are exploring the roles of remodeling complexes that are capable of nucleosome mobilization, such as SWI/SNF, in disruption of chromatin structure. We have written a review of our work, placing it in the context of the chromatin field (Cole et al., 2012a) and we have described our methodology in detail (Cole et al., 2012b). Cole HA, Howard BH, Clark DJ (2011a). The centromeric nucleosome of budding yeast is perfectly positioned and covers the entire centromere. Proc Natl Acad Sci USA 108, 12687-12692. Cole HA, Howard BH, Clark DJ (2011b). Activation-induced disruption of nucleosome position clusters on the coding regions of Gcn4-dependent genes extends into neighbouring genes. Nucl Acids Res 39, 9521-9535. Cole HA, Nagarajavel V, Clark DJ (2012a). Perfect and imperfect nucleosome positioning in yeast. Biochim Biophys Acta 1819, 639-643. Cole HA, Nagarajavel V, Clark DJ (2012b). Genome-wide mapping of nucleosomes in yeast using paired-end sequencing. Methods in Enzymology 513, 145-168. 2. SPT10 AND SBF CONTROL THE TIMING OF HISTONE H2A/H2B GENE ACTIVATION IN BUDDING YEAST. We have shown that Spt10 is a very unusual trans-activator, in which a HAT domain, normally recruited as a co-activator to promoters through an activation domain, is attached directly to a sequence-specific DNA-binding domain. More recently, we have addressed the role of Spt10 in the cell cycle-dependent regulation of the histone genes, which is necessary to provide histones for nucleosome assembly during DNA replication (Eriksson et al., 2011). Histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. We showed that Spt10 binds to two pairs of UAS elements in the HTA1-HTB1 promoter: UAS1/UAS2 drive HTA1 expression and UAS3/UAS4 drive HTB1. UAS3 and UAS4 also contain binding sites for the cell cycle regulator SBF (a Swi4-Swi6 heterodimer), which overlap the Spt10 binding sites. Both SBF and Spt10 are bound in cells arrested with alpha-factor, apparently awaiting a signal to activate transcription. Soon after removal of alpha-factor, SBF initiates a small, early peak of HTA1 and HTB1 transcription, which is followed by a much larger peak due to Spt10. Both activators dissociate from the HTA1-HTB1 promoter after expression has been activated. Thus, SBF and Spt10 cooperate to control the timing of HTA1-HTB1 expression (Eriksson et al., 2011). Our current work has two goals: (1) Understanding the dynamics of chromatin structure during the cell cycle at the histone genes and genome-wide. (2) Testing our proposed model for histone gene regulation (Eriksson et al., 2012), which posits that the extent to which histone chaperones are saturated with histones is the critical signal for negative feedback control of transcription. Eriksson PR, Ganguli D, Clark DJ (2011). Spt10 and Swi4 control the timing of histone H2A/H2B gene activation in budding yeast. Mol Cell Biol 31, 557-572. Eriksson PR, Ganguli D, Nagarajavel V, Clark DJ (2012). Regulation of histone gene expression in budding yeast. Genetics 191, 7-20. 3. STRUCTURE OF THE C-TERMINAL DOMAIN OF THE LINKER HISTONE, H1. In collaboration with Dr. Jeffrey Hayes, University of Rochester Medical Center. The structure of the C-terminal domain of histone H1 has been a long-term interest of mine. We showed that this domain undergoes a transition from intrinsic disorder to a folded structure when binding to nucleosomal DNA (Fang et al., 2012). All of the experimental work was done in the Hayes Lab. Fang H, Clark DJ, Hayes JJ (2012). DNA and nucleosomes direct distinct folding of a linker histone H1 C-terminal domain. Nucleic Acids Res 40, 1475-1484.